Abstract: Natural killer (NK) cells are innate lymphocytes that express germline encoded activating and inhibitory receptors. These cells have the capacity to rapidly kill virus- infected cells and cancer cells and modulate adaptive immune responses by regulating dendritic cells (DCs) and through production of pro-inflammatory cytokines. NK cells develop in the bone marrow and thymus from progenitors that also give rise to B and T lymphocytes, respectively. Despite the severe consequences of failed NK cell development (immune deficiency and malignancy), little is known about the mechanisms that control specification and commitment to this lymphoid lineage. While multiple transcription factors are required for NK cell development and function, most are required at a relatively late stage in the differentiation process and none of these factors are NK cell lineage specific. Therefore, the transcriptional networks that specify activation of the NK cell gene program from multipotent progenitors and maintain commitment to the NK cell lineage remain to be elucidated. We have focused on determining the functions of the E proteins and their antagonists, the inhibitors of differentiation (Id) proteins, since these transcription factors appear to function as molecular switches directing B or T versus NK cell differentiation, respectively. In Aim 1 of this grant application we propose experiments to test the hypothesis that Id2 and Id3 cooperate to promote NK cell development in the bone marrow and thymus. In Aim 2 we will determine the mechanisms by which E proteins antagonize NK cell development in different microenvironments. We will test the hypothesis that aberrant E protein activity results in activation of the B or T cell gene programs thereby causing lineage diversion or lineage confusion in NK lineage cells. In Aim 3 we will characterize the transcription factors that promote NK cell lineage specific gene expression and determine whether these factors play a direct role in NK cell lineage specification. By identifying the essential components of the NK cell transcriptional program, and how these factors are influenced by the microenvironment, we will provide a basis for understanding how commitment to the NK cell lineage is achieved. Moreover, these studies will provide a foundation for future therapies aimed at altering the outcome of NK cell differentiation and for predicting the effects of therapeutic interventions on this developmental program. Project Narrative: Natural killer (NK) cells are lymphocytes that function in both adaptive and innate immune responses. The goal of our research is to determine the mechanisms controlling development NK cells from multipotent progenitors by identifying the essential transcription factors that promote lineage specification and commitment. Our studies will provide insight into how to how to manipulate this program to avoid disease and how therapeutic interventions will impact on NK cell development.